Polyketides are a large, structurally diverse family of natural products. Polyketides possess a broad range of biological activities including antibiotic and pharmacological properties. For example, polyketides are represented by such antibiotics as tetracyclines and erythromycin; anticancer agents including daunomycin; immunosuppressants, for example FK506 and rapamycin; veterinary products such as monensin and avermectin; and agriculturally used compounds such as spinocyn (insecticidal) and soraphen (antifungal). Polyketides are especially abundant in a class of mycelial bacteria, the actinomycetes.
Polyketide synthases are multifunctional enzymes related to fatty acid synthases (FASs). PKS catalyze the biosynthesis of polyketides through repeated (decarboxylative) Claisen condensations between acylthioesters, usually acetyl, propionyl, malonyl or methylmalonyl. Following each condensation, they introduce structural variability into the product by catalyzing all, part, or none of a reductive cycle comprising a ketoreduction, dehydration, and enoylreduction on the .beta.-keto group of the growing polyketide chain. After the carbon chain has grown to a length characteristic of each specific product, it is released from the synthase by thiolysis or acyltransfer. Thus, PKS consist of families of enzymes which work together to produce a given polyketide. It is the controlled variation in chain length, choice of chain-building units, and the reductive cycle, genetically programmed into each PKS, that contributes to the variation seen among naturally occurring polyketides. The polyketides resulting from the reactions catalyzed by the PKS often require further modification, such as glycosylation, in order to provide antibiotic activity.
Three general classes of PKS exist. One class, known as Type I, "complex" or "modular" PKS, is represented by the PKS for macrolides such as erythromycin. The "modular" PKS are assemblies of several large multifunctional proteins carrying, between them, a set of separate active sites for each step of carbon chain assembly and modification (Cortes, J., et al., Nature (1990) 348:176; Donadio, S., et al., Science (1991) 252:675; MacNeil, D. J., et al., Gene (1992) 115:119). Structural diversity occurs in this class from variations in the number and type of active sites in the PKS. This class of PKS displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone. (See FIG. 1.)
The second class of PKS, called Type II or "aromatic" PKS, is represented by the synthases for aromatic compounds. The "aromatic" PKS are typically encoded by at least three separate open reading frames and have a single set of iteratively used active sites (Bibb, M. J., et al., EMBO J. (1989) 8:2727; Sherman, D. H., et al., EMBO J. (1989) 8:2717; Femandez-Moreno, M. A., et al., J. Biol. Chem. (1992) 267:19278). (See FIG. 2.)
A third class of PKS is generally known as "fungal" PKS and is a multifunctional protein encoded in a single reading frame. A typical "fungal" PKS is 6-methyl salicylic acid synthase (MSAS) characterized from Penicillium patulum. The gene has also been isolated from Aspergillus nidulans and from Colletotrichum lagenarium and a PKS having norsolorinic acid as a product from A. nidulans. Fujii, I., et al., Mole Gen Genet (1996) 253:1-10. The fungal PKS thus do not fit neatly into the categorization of aromatic versus modular and thus constitute a third group. (See FIG. 3.)
Streptomyces is an actinomycete which is an abundant producer of aromatic polyketides. In each Streptomyces aromatic PKS so far studied, carbon chain assembly requires the products of three open reading frames (ORFs). ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT) active site; ORF2 encodes a protein similar to the ORF1 product but lacking the KS and AT motifs; and ORF3 encodes a discrete acyl carrier protein (ACP).
For example, Streptomyces coelicolor produces the blue-pigmented polyketide, actinorhodin. The actinorhodin gene cluster (act), has been cloned. The cluster encodes the PKS enzymes described above, a cyclase and a series of tailoring enzymes involved in subsequent modification reactions leading to actinorhodin, as well as proteins involved in export of the antibiotic and at least one protein that specifically activates transcription of the gene cluster. Other genes required for global regulation of antibiotic biosynthesis, as well as for the supply of starter (acetyl CoA) and extender (malonyl CoA) units for polyketide biosynthesis, are located elsewhere in the genome. The act gene cluster from S. coelicolor has been used to produce actinorhodin in S. parvulus. Malpartida, F., et al., Nature (1984) 309:462.
Bartel, et al., J Bacteriol (1990) 172:4816-4826, recombinantly produced aloesaponarin II using S. galilaeus transformed with an S. coelicolor act gene cluster consisting of four genetic loci, actI, actIII, actIV and actVII. Hybrid PKS, including the basic act gene set but with ACP genes derived from granaticin, oxytetracycline, tetracenomycin and frenolicin PKS, have also been designed which are able to express functional synthases. Khosla, C., et al., J Bacteriol (1993) 175:2197-2204. Hopwood, D. A., et al., Nature (1985) 314:642-644, describes the production of hybrid polyketides, using recombinant techniques. Sherman, D. H., et al., J Bacteriol. (1992) 174:6184-6190, reports the transformation of various S. coelicolor mutants, lacking different components of the act PKS gene cluster, with the corresponding granaticin (gra) genes from S. violaceoruber, in trans.
Recombinant production of heterologous functional PKS--i.e., a PKS which is capable of producing a polyketide--has been achieved in Streptomyces and hybrid forms of aromatic PKS have been produced in these hosts as well. See, for example, Khosla, C., et al., J Bacteriol (1993) 175:2194-2204; Hopwood, D. A., et al., Nature (1985) 314:642-644; Sherman, D. H., et al., J Bacteriol (1992) 174:6184-6190. In addition, recombinant production of modular PKS enzymes has been achieved in Streptomyces as described in PCT application W095/08548. However, a single vector which carried genes encoding PKS catalytic sites was transformed into E. coli by Roberts, G. A., et al., Eur J Biochem (1993) 214:305-311, but the PKS was not functional, presumably due to lack of Phospho pantetheinylation of the acyl carrier proteins.
Recombinant production of functional polyketide synthases in Streptomyces hosts was also described in PCT applications W098/01546 and W098/01571, both published Jan. 15, 1998.
A large number of polyketide synthases have been cloned, including the PKS for the production of avermectin (U.S. Pat. No. 5,252,474); spiramycin (U.S. Pat. No. 5,098,837); and tylosin (European application publication no. 791,655 published Feb. 19, 1997).
U.S. Pat. No. 5,716,849 describes the recovery and sequencing of the nucleotide sequence encoding the PKS cluster for the production of soraphen. The disclosure, which is incorporated herein by reference, prophetically describes the expression of the soraphen PKS encoding nucleotide sequence in bacteria, yeast and plants. The disclosure indicates that the PKS proteins produced will be functional in synthesis of polyketides. However, actual expression and functionality were not demonstrated.
It is known that in order for the PKS cluster to be functional, the translated apo-PKS must be phosphopantetheinylated enzymatically to obtain holo-ACP synthase components. Carreras, C.W., et al., Biochemistry (1997) 36:11757-11761. The conversion from apo-PKS to PKS containing holo-ACPs requires an appropriate phosphopantetheinyl transferase (PPT). It has been recognized that PPT enzymes are members of a superfamily, but they are not interchangeable. For example, PPT enzymes which are able to convert apo-fatty acid synthase enzyme clusters to the holo-form are not effective in thus converting typical PKS apoenzymes. Lambalot, R. H., et al., Chem and Biol (1996) 3:923-936. The necessity for providing PPT activity in order to obtain functional PKS recombinantly in E. coli was described by Cox, R. J., et al., FEBS LETT (1997) 405:267-272. In addition, WO 98/27203, the contents of which are incorporated herein by reference, demonstrates the recombinant production of a functional 6-MSAS in yeast using a fusion protein containing the 6-MSAS from P. patulum and the PPT enzyme (holo-ACP synthase) from Bacillus subtilis, specifically surfactant holo-ACP synthase (Sfp). Production of 6-MSA in E. coli modified to contain an expression system for 6-MSAS, along with an expression system for the Sfp gene was also demonstrated under specified conditions.
The present invention provides plants transformed to contain a single or multivector system for the production of PKS and the resultant polyketides. The use of plants for the expression and generation of functional PKS allows a suitable source for large scale production of PKS and polyketides; in addition, the polyketides produced may confer desirable properties on the plants producing them, such as insect resistance.